Illumina HiSeq 4000 paired end sequencing; Exome-capture RNAseq of matched microdissected FF and FFPE human breast cancer cells, cancer-associated fibroblasts, and surrounding tumor-infiltrating lymphocytes.
Exome-capture RNAseq of matched microdissected FF and FFPE human breast cancer cells, cancer-associated fibroblasts, and surrounding tumor-infiltrating lymphocytes.
For comparison between fixed and frozen tissue transcriptomics, matched FFPE and fresh frozen samples of TNBC were provided from the division of clinical pathology, Hôpitaux Universitaires de Genève. The research protocol was implemented in accordance with the declaration of Helsinki and Swiss law on biomedical research and was approved by the ethics committee of Geneva (CCER: 2018-02333).     FFPE tissue sections were cut, mounted on a polyethylene terephthalate membranes (PET) slide and stained with hematoxylin followed by Laser capture microdissection. Briefly, metal frame slides mounted with PET (Leica, Mannheim, Germany) were pretreated by coating with poly-lysine (Sigma-Aldrich, St-Louis, United States) and exposure to UV (15 mW/cm2/s) for 30 min. Consecutive sections from FFPE blocks were cut just before microdissection at 8 μm, floated onto a 45°C water bath and mounted on PET slides. After drying for 30 min, tissue sections were deparaffinized in xylene, rehydrated with decreasing concentrations of ethanol, stained with hematoxylin, dehydrated with increasing concentrations of ethanol, and cleared in xylene.     Fresh frozen tissue sections were microdissected with few differences in sample preparation compared to FFPE samples. Briefly, metal frame slides mounted with PET (Leica, Mannheim, Germany) were exposed to UV (15 mW/cm2/s) for 30 min and pre-cooled at -20°C for 1h. Consecutive sections from fresh frozen blocks were cut at 8 μm in a cryostat and mounted on cold PET slides just before microdissection. Tissue sections were fixed in ethanol, stained with freshly prepared cresyl violet and cleared in ethanol. Each fresh frozen slide was microdissected within 20min after staining.     For both  FFPE and fresh frozen tissue, a minimal surface of 2.5 mm2 bulk tissue was microdissected using the Laser Microdissection Systems Leica LMD7000 and collected in 0.5 mL tubes. Laser parameters were set as follows: laser power of 39 mW, a wavelength of 349 nm, pulse frequency of 664 Hz, pulse energy of 120 μJ. Quality control was performed on the raw sequencing data using the FastQC tool (20)20. Andrews S. FastQC: a quality control tool for high throughput sequence data 2010 [Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Microdissected cells were lysed by incubation with proteinase K at 56°C and stirring at 300 rpm. RNA was extracted in an RNAse-free environment using Qiagen® RNeasy FFPE kit (Qiagen, Hilden, Germany) according to the supplier's protocol, treated with DNase I, eluted in 15 µl of RNAse-free water and stored at -80°C. Total RNA was quantified using Qubit™ RNA HS Assay Kit and Qubit® Fluorometer (Thermo Fisher Scientific, MA, USA). RNA fragment size was analyzed with the 2100 Bioanalyzer (Agilent, CA, USA) using Eukaryote total RNA Pico assay (Agilent, CA, USA). Libraries were prepared using TruSeq RNA Exome® kit (Illumina Inc, CA, USA) according to manufacturer's protocols. The input quantities of RNAs were adjusted to the DV200 values as recommended in the TruSeq RNA Exome® reference guide and technical note (No. 470-2014-001): 40 ng if DV200 is between 50%-70% and 55 ng if DV200 is between 30%-50%. 3 out of 16 RNA samples were concentrated using a Vacuum Concentrator Centrifuge (UNIVAPO 100 ECH, Uniequip, Germany) to reach the minimal volume recommended by the TruSeq RNA Exome® kit.